Although bacterial ribosomes are a popular target of common antibiotics, there are no drugs that specifically target ribosome assembly. To date, there is simply no screening method that would be suitable for assaying inhibitors of ribosomal subunit assembly. Prof. Dr. Elke Deuerling and Dr. Rainer Nikolay from the University of Konstanz have now achieved a breakthrough. They have developed a method that enables the high-throughput identification of inhibitors of ribosome biogenesis. The method has the potential of being used for the development of antibiotics that effectively kill antibiotic-resistant bacteria.
Ribosomes are the site where cellular translation takes place and play a central role in the biosynthesis of proteins. As ribosomes from bacteria and higher organisms differ significantly in their structure, these differences allow certain drugs to inhibit bacterial ribosomes while leaving human ribosomes unaffected. This makes bacterial ribosomes ideal targets for antibiotics. “There are many antibiotics that target bacterial ribosomes; most of them interfere with the translation machinery and hence with protein biosynthesis,” explains Elke Deuerling, professor of molecular microbiology at the University of Konstanz. Deuerling’s research is specifically focused on ribosomes.
In addition to focusing on the function of ribosomes, experts have long considered the ribosomes’ complex assembly an attractive target for novel antimicrobial drugs. The biogenesis of ribosomes is a process that requires the precise spatial and temporal coordination of many factors. Ribosomal RNAs and ribosomal proteins need to be assembled in a precisely coordinated process into two huge subunits. This involves so-called assembly factors that ensure that the subunits are assembled in an orderly and efficient way. “In theory, every single assembly step can be inhibited, either directly or indirectly, by way of specific assembly factors,” explains Deuerling.
Nevertheless, there are no antibiotics on the market that specifically inhibit ribosome assembly. “Quite simply, we lack a screening concept that would allow us to specifically identify such substances,” says Dr. Rainer Nikolay, a scientist in Prof. Deuerling’s laboratory.
During his post-doc period in the pharmaceutical industry, Nikolay gained comprehensive experience in the development of assay systems. He then sought to put this experience to good use in the design of an assay focusing specifically on ribosome function. When Nikolay joined Deuerling’s laboratory in 2010, he came up with the idea of studying ribosome assembly using fluorescent markers. The approach was based on bacterial reporter strains with ribosomes whose two subunits carried different fluorescent labels – one subunit with a red fluorescent label and the second with a green one.
In order to do this, genes that code for fluorescent proteins were fused with genes that code for proteins of the small or large ribosomal subunit. “The decisive factors were the selection of the appropriate ribosomal genes, followed by the careful examination of the reporter strains generated in order to prevent unwanted side effects resulting from ribosomal manipulation,” explains Nikolay. He successfully constructed reporter strains whose growth properties were almost identical to those of the wild-type strain and in which the assembly and activity of the translation apparatus were not affected.
The reporter strains were then used to study the biogenesis of ribosomes in more detail. “Both ribosomal subunits are naturally produced in a certain ratio and they are assembled in a highly complex and strictly regulated process,” explains Deuerling. As each subunit carried a fluorescent label, the researchers were able to determine the ratio of red to green fluorescence in the presence of the substance to be tested and compare the ratio with that in untreated bacterial strains. The red:green ratio provided the researchers with information as to whether the biogenesis of the two subunits was proceeding as normal or whether the assembly of one of the two subunits was defective.
Once the functionality of the method had been validated in the laboratory, the procedure was adapted to a format suitable for use in high-throughput analyses. This involved the optimisation and standardisation of the method for use with microtitre plates and automated screening procedures. High-throughput screening (HTS) can thus be applied to identify compounds with the potential to inhibit ribosome assembly.
The compounds identified are ideal candidates for antibiotics development. “Ribosomes are the protein builders and proteins cannot be produced without ribosomes. The effect of inhibited ribosome assembly translates directly into the inhibition of bacterial growth,” says Deuerling. Bacteria that fail to produce functional ribosomes die as all vital functions come to a standstill. “In order to prevent bacteria from becoming resistant to a specific compound, several ribosome assembly inhibitors can be combined,” says Deuerling highlighting the advantages of their approach.
In order to bring the patent-pending screening method to application, Deuerling and her team, supported by colleagues from the Departmetn of Chemical Biology and the Screening Center at the University of Konstanz, are carrying out the initial screen themselves. “This helps us to compare on a small scale our new method with methods used in the pharmaceutical industry and with a bit of luck we might even manage to identify interesting assembly inhibitors,” says Nikolay. Deuerling and her team are very interested in contacting companies and investors who may be able to support them in the further development and commercialisation of their screening approach. “I am sure that in the future we will need more partners that can help us transfer the method from bench- to bedside,” says Deuerling.
In addition to enabling the targeted search for new antibacterial compounds, the screening approach also opens up new applications in basic research, for example for the scientific investigation of ribosome assembly. “Chemical substances that we will identify using our screening approach might be used to answer unresolved questions related to the biogenesis of ribosomes,” says Deuerling. The approach has the potential for further refinement in order to visualize individual steps of the ribosome assembly process. “We believe that our innovative method has a big potential,” concludes Deuerling.
Further information:Prof. Dr. Elke DeuerlingDepartment of Molecular MicrobiologyFaculty of BiologyUniversity of KonstanzTel.: +49 (0)7531/882647E-mail: elke.deuerling(at)uni-konstanz.de